CN117500534A

CN117500534A - Hyaluronic acid crosslinked body and filler composition comprising same

Info

Publication number: CN117500534A
Application number: CN202280043086.9A
Authority: CN
Inventors: 林千洙; 梁钟铁; 李彰训
Original assignee: Medy Tox Inc
Current assignee: Medy Tox Inc
Priority date: 2021-06-17
Filing date: 2022-06-15
Publication date: 2024-02-02
Also published as: WO2022265380A1; EP4356935A1; KR20220168990A

Abstract

The present invention relates to a hyaluronic acid crosslinked body and a filler composition including the same, and more particularly, to a hyaluronic acid crosslinked body having high cohesion and adhesion while reducing swelling phenomenon, and a filler composition including the same.

Description

Hyaluronic acid crosslinked body and filler composition comprising same

Technical Field

The present disclosure relates to a hyaluronic acid crosslinked body and a filler composition including the same, and more particularly, to a hyaluronic acid crosslinked body having high cohesion and adhesion while reducing swelling phenomenon, and a filler composition including the same.

Background

Hyaluronic Acid (HA) is typically a linear polysaccharide with a high average molecular weight. Hyaluronic acid has a negative charge as a polymer of D-glucuronic acid and N-acetyl-D-glucosamine. Hyaluronic acid is found mainly in the extracellular and intercellular stroma, but is also present in the cell. As described above, hyaluronic acid is a substance existing in the body, and thus has biocompatibility, and also has a characteristic that crosslinked hyaluronic acid hydrogel can be easily prepared by a crosslinking agent or the like. Thus, since the first release of the rayleigh (Restylane) product in the 90 th 20 th century (galderm), the filler (filer) product using crosslinked hyaluronic acid has been widely used worldwide.

The hyaluronic acid gels currently used as fillers are prepared by cross-linking natural hyaluronic acid acids into a network by a cross-linking reaction. Natural hyaluronic acid and certain partially cross-linked hyaluronic acid absorb water until it is completely dissolved in water, but cross-linked hyaluronic acid gels typically absorb a certain amount of water until they are saturated, thereby having a limited degree of swelling (swelling).

On the other hand, the filler products using the crosslinked hyaluronic acid gel may be classified as products having a single phase or two phases. The viscosity modulus (visual modulus) of the single-phase crosslinked hyaluronic acid gel is high and the Elastic modulus (Elastic modulus) is low. The biphasic crosslinked hyaluronic acid gel has a low viscous modulus and a high elastic modulus. The monophasic crosslinked hyaluronic acid gel and the biphasic crosslinked hyaluronic acid gel also have different properties in vivo. For example, a single-phase crosslinked hyaluronic acid gel having a low elastic modulus and a high viscosity modulus is excellent in cohesive force, and thus has a low possibility of detachment from an injection site, and has a characteristic of being additionally water-absorbable, and thus has a characteristic of continuously absorbing ambient moisture also in vivo. Therefore, the single-phase crosslinked hyaluronic acid gel generally cannot maintain the original injection form at an early stage after injection into the body, and has a disadvantage of an increase in volume over the original injection. In contrast, a biphasic crosslinked hyaluronic acid gel having a high elastic modulus but a low viscous modulus can maintain an injection form for a long period of time with a small volume change, but has a characteristic of being easily detached from an injection site or not being uniformly distributed to the injection site.

On the other hand, a swelling phenomenon may occur after injection of the hyaluronic acid filler, which is a phenomenon in which the injection site and the peripheral site swell due to absorption of body fluid from peripheral tissues of the injection site by the hyaluronic acid gel composition injected into the body. This swelling phenomenon is a side effect common in the injection of fillers from the point of swelling at the injection site, which is an undesirable phenomenon in terms of the viscosity and elasticity of the hyaluronic acid gel and the decrease in strength due to absorbed water and thus the decrease in the injection effect of the fillers.

Thus, there remains a need for a hyaluronic acid filler that maintains adequate viscoelasticity and gel strength while having less swelling when injected into the body.

Disclosure of Invention

Technical problem

The present disclosure aims to provide a crosslinked hyaluronic acid having a critical strain (critical strain) of 10% to 100% and a swelling degree (swelling degree) of 1% to 100%.

Another object of the present disclosure is to provide a filler composition including the hyaluronic acid cross-linked body.

Technical proposal

In one aspect, the present disclosure provides a crosslinked hyaluronic acid having a critical strain of 10% to 100% and a swelling degree of 1% to 100%.

In particular embodiments, the average particle size of the hyaluronic acid cross-links may be in the range of 50 μm to 150 μm.

In a specific embodiment, the elastic modulus (G') of the hyaluronic acid cross-linked body may be 400Pa to 2000Pa.

In a specific embodiment, the viscous modulus (G') of the hyaluronic acid cross-linked body may be 100Pa to 600Pa.

In a specific embodiment, the compressive force of the hyaluronic acid cross-linked body may be 15n·s to 100n·s.

In a specific embodiment, the injection force of the hyaluronic acid cross-linked body may be 1N to 50N.

In particular embodiments, the hyaluronic acid crosslinks may be crosslinked by a crosslinker having difunctional epoxy groups. The crosslinking agent having a difunctional epoxy group may be at least one selected from the group consisting of 1, 4-butanediol diglycidyl ether, polyethylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, polytetramethylene glycol diglycidyl ether, polyglycerol polyglycidyl diether, glycerol diglycidyl ether, triethylenediglycidyl ether, trimethylolpropane triglycidyl ether, ethylene diglycidyl ether, neopentyl glycol diglycidyl ether, and 1, 6-hexanediol diglycidyl ether.

Another aspect of the present disclosure provides a filler composition comprising the hyaluronic acid cross-linked body.

In particular embodiments, the concentration of hyaluronic acid crosslinker in the bulking agent composition can be 10mg/mL to 30mg/mL.

In particular embodiments, the filler composition may not further include uncrosslinked hyaluronic acid.

In particular embodiments, the filler composition may further comprise a local anesthetic.

In particular embodiments, the filler composition may be filled in a syringe.

In particular embodiments, the filler composition may be used for at least one use selected from the group consisting of facial reshaping, wrinkle improvement, facial contour surgery, chest reshaping, breast augmentation, genital enlargement, glans augmentation, urinary incontinence treatment, and arthritis treatment.

Advantageous effects

The crosslinked hyaluronic acid gel according to the present disclosure is characterized by having high cohesion and high adhesion, and thus maintaining integrity without detachment from peripheral portions, while swelling phenomenon after injection is reduced.

Drawings

Fig. 1 is a graph showing the change in volume of hyaluronic acid gel injected after injecting a cross-linked hyaluronic acid sample into a nude mouse over time.

Best mode

The present disclosure provides a hyaluronic acid crosslinker, which generally has a critical strain of 10% to 100% and a swelling degree of 1% to 100%.

In the present specification, the term "hyaluronic acid" refers to hyaluronic acid (hyaluronic acid) having a chemical formula in formula 1, sodium hyaluronate (hyaluronate), or a pharmaceutically acceptable salt thereof.

In formula 1, n is the number of repeating units. The hyaluronic acid may be of any origin. For example, hyaluronic acid may be of animal or non-animal origin. The hyaluronic acid may be derived from bacteria. The bacteria may be derived from the genus streptococcus. The streptococcus bacteria may be streptococcus equi (Streptococcus equi), streptococcus pyogenes(s) or streptococcus zooepidemicus(s). The hyaluronic acid may be commercially available.

The molecular weight of the hyaluronic acid may be 1000000Da to 2500000Da, for example 1000000Da to 2000000Da, 1500000Da to 2500000Da, or 1500000Da to 2000000Da.

The intrinsic viscosity (Intrinsic Viscosity) of the hyaluronic acid may be 1.0m ³ Kg to 5.0m ³ Kg, e.g. 1.5m ³ Kg to 4.0m ³ /kg、2.5m ³ Kg to 4.0m ³ /kg、2.5m ³ Kg to 3.5m ³ Per kg, or 2.5m ³ Kg to 3.0m ³ /kg。

In the present specification, the term "hyaluronic acid cross-linked body" refers to cross-linked hyaluronic acid, which is used interchangeably with "hyaluronic acid cross-linked body", "cross-linked hyaluronic acid".

In the present specification, the term "swelling" refers to a phenomenon in which a crosslinked hyaluronic acid gel swells after absorbing water.

In the present specification, the term "swelling degree" means a weight ratio of the crosslinked hyaluronic acid gel completely swelled when the crosslinked hyaluronic acid is immersed in water, compared to before swelling. The swelling degree can be obtained, for example, by the following calculation formula described in the measurement method.

In the present specification, the critical strain is a detection index for confirming a strain value required to destroy an internal structure of a substance including linear viscoelastic properties when shear deformation is applied to the substance. In general, a crosslinked hyaluronic acid gel is composed of a crosslinked hyaluronic acid gel or of a crosslinked hyaluronic acid gel and a solution, and its interior is subjected to various internal interaction forces caused by hydrogen bonds, covalent bonds, van der Waals bonds, physical bonds, and the like. Internal interactions can be classified as gel-gel, gel-solution, or solution-solution interactions, with the solution having a higher molecular mobility than the gel and a smaller area than the gel, and thus the size of the interactions can be considered as gel-gel > gel-solution > solution-solution. Taking the example of a single phase in gel-gel form alone, it has a higher interaction, and thus it can be predicted that its internal structure will be destroyed in a higher strain value. In contrast, since the two phases have a form in which gel and solution coexist, gel-gel interactions are reduced, and instead gel-solution interactions and solution-solution interactions are further enhanced, it can be predicted that the sum of interactions of the internal structures thereof becomes smaller than that of the single phase, and thus that the internal structures thereof are broken in a smaller strain value.

Furthermore, it is predicted that a general single-phase filler product will have a characteristic of stronger cohesion due to a similar phenomenon, and that a dual-phase filler product exhibits a relatively smaller cohesion than a single-phase filler. Cohesive force characteristics are characteristics that can be used as an index for predicting the extent of adhesion of an injectate in vivo. To verify this, it was confirmed that the monophasic product exhibits a higher adhesion value than the biphasic product by comparing the adhesion value, which is an index for measuring the degree of cohesion of the biphasic filler product and the monophasic filler product.

The hyaluronic acid cross-linked according to an aspect of the present disclosure has a high critical strain as the existing single-phase hyaluronic acid fillers, in contrast to unexpected characteristics at the point of low swelling degree. In addition, the hyaluronic acid crosslinked body of the present disclosure is characterized in that it has high cohesion and high adhesion to a degree similar to those of the existing single phase fillers, and thus is not easily detached from an injection site, and simultaneously can maintain an injection form for a long period of time due to a high elastic modulus (G').

The (i) critical strain of the hyaluronic acid cross-linked body according to the present disclosure may be 10% to 100%, such as 10% to 50%, 10% to 30%, or 10% to 40%, such as 10% to 50%, such as 10% to 30%, and the swelling degree may be 1% to 100%, such as 1% to 90%, 1% to 80%, 1% to 70%, 1% to 60%, 1% to 50%, 10% to 60%, 20% to 60%, 30% to 60%, 40% to 50%, and 50% to 60%.

The average particle size (um) of (ii) of the hyaluronic acid cross-linked body may be 50um to 150um, for example 60um to 150um, 70um to 150um, 80um to 150um, 90um to 150um,60 um to 140 um,60 um to 130 um,60 um to 120 um, and 60um to 100 um.

The elastic modulus (G') of (iii) of the hyaluronic acid cross-linked body may be 400Pa to 2000Pa, for example 500Pa to 1900Pa, 600Pa to 1800Pa, or 600Pa to 1700Pa.

The (iv) viscous modulus (G ") of the hyaluronic acid cross-linked body may be 100Pa to 600Pa, for example 120Pa to 500Pa, 150Pa to 450Pa, or 160Pa to 350Pa.

The (v) compressive force of the hyaluronic acid cross-linked body may be 15 n.s to 100 n.s, for example 20 n.s to 90 n.s, 25 n.s to 80 n.s, or 30 n.s to 75 n.s.

The (vi) injection force of the hyaluronic acid cross-linked may be 1N to 50N, for example 3N to 45N, 4N to 40N, or 7N to 35N.

In a specific embodiment, the hyaluronic acid cross-linked body satisfies one, two, three, four or five of the conditions (ii) to (vi) while satisfying the above condition (i).

The hyaluronic acid cross-linked body according to the present disclosure may be manufactured by a process including, for example, the following steps, but is not limited thereto:

(a) Crosslinking and bonding are performed by adding a crosslinking agent to hyaluronic acid dissolved by an alkaline aqueous solution;

(b) Dialysis, washing and/or swelling of the crosslinked gel;

(c) Crushing; and

(d) Filled into the syringe.

In the above process steps, the order of steps (b) to (d) may be changed or repeated two or more times. For example, the comminution step may be carried out in addition to step (c) between steps (a) and (b), or as an end in step (c), or instead of step (c) only in steps (a) and (b).

In addition, in addition to the above steps (a) to (d), other steps may be further included, for example, a step of adding an anesthetic agent as described below, and/or a step of performing high-temperature steam sterilization. For example, the step of adding an anesthetic may be added between steps (b) and (c), or between steps (c) and (d). The step of performing high temperature steam sterilization may be added between steps (c) and (d), or after step (d).

In the above step (a), the alkaline aqueous solution may be NaOH, KOH, and preferably may be NaOH aqueous solution, but is not limited thereto. When aqueous NaOH is used, the concentration may be 0.1% (w/w) to 2.5% (w/w), for example 0.1% (w/w) to 2.0% (w/w), or 0.5% (w/w) to 1.0% (w/w).

In addition, the concentration of hyaluronic acid dissolved in the alkaline aqueous solution may be 10% (w/w) to 25% (w/w), for example 10% (w/w) to 20% (w/w) or 10% (w/w) to 15% (w/w).

In step (a), the crosslinking agent may have a multifunctional group. The crosslinker may have difunctional epoxy groups. The crosslinking agent having a difunctional epoxy group may be at least one selected from the group consisting of 1, 4-butanediol diglycidyl ether, polyethylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, polytetramethylene glycol diglycidyl ether, polyglycerol polyglycidyl diether, glycerol diglycidyl ether, triethylenediglycidyl ether, trimethylolpropane triglycidyl ether, ethylene diglycidyl ether, neopentyl glycol diglycidyl ether, and 1, 6-hexanediol diglycidyl ether.

Further, the crosslinking reaction may be performed at normal temperature or higher, for example, 20 ℃ to 40 ℃, 25 ℃ to 40 ℃, or 30 ℃ to 40 ℃, and the reaction time may be 24 hours or less, for example, 10 hours to 24 hours, 15 hours to 24 hours, or 15 hours to 20 hours.

In the above step (b), dialysis refers to a step of mixing a dialysis membrane or gel crosslinked in a dialysis container with a dialysis solution comprising a certain buffer solution (e.g., naCl solution, KOH solution, PBS solution, etc.), then performing dialysis, washing, and swelling the dialyzed gel. The series of dialysis, washing and expansion steps described above may be repeated two or more times. The unreacted cross-linking agent is removed and/or removed after passing through the series of dialysis, washing and swelling steps described above, and the pH and/or osmotic pressure of the cross-linked gel may be adjusted.

In the step (c), the pulverization may be performed at 1000rp to 10000rpm, for example, 2000rpm to 9000rpm, 3000rpm to 8000rpm, 4000rpm to 7000rpm, 5000rpm to 9000rpm, or 5000rpm to 8000rpm for 60 seconds or more, for example, 60 seconds to 100 seconds, 70 seconds to 120 seconds, 80 seconds to 140 seconds, 90 seconds to 160 seconds, 100 seconds to 180 seconds, 120 seconds to 240 seconds, using a pulverizer.

In another aspect, a filler composition comprising the hyaluronic acid cross-linked body is provided.

In the composition, the final concentration of the crosslinked hyaluronic acid gel may be greater than or equal to 10mg/ml, greater than or equal to 15mg/ml, greater than or equal to 18mg/ml, or greater than or equal to 19mg/ml. For example, the concentration of the crosslinked hyaluronic acid may be from 10mg/ml to 30mg/ml, from 12mg/ml to 28mg/ml, from 14mg/ml to 26mg/ml, from 16mg/ml to 24mg/ml, from 16mg/ml to 20mg/ml, or about 18mg/ml.

The composition may not further include uncrosslinked hyaluronic acid. The term "further includes" uncrosslinked hyaluronic acid means that uncrosslinked hyaluronic acid is artificially further added to the hyaluronic acid crosslinked body, and the term "not further includes uncrosslinked hyaluronic acid means that uncrosslinked hyaluronic acid is artificially further added to the hyaluronic acid crosslinked body.

The composition may further comprise an anesthetic. The anesthetic may be a local anesthetic. The anesthetic may be selected from the group consisting of ambucine (ambucaine), amolanone (amantane), isovaleric acid (amoxicaine), butoxyprocaine (benoxinate), benzocaine (benzocaine), betaxocaine (betaxycaine), phenylsulfane (biphenamine), bupivacaine (bupivacaine), aminobutyric acid (bupropion), bupivacaine (bupropione), bupivacaine (bupivacaine), ding Tuoxi-caine (bunaxycaine), carbocaine (carbetaine), chloroprocaine (chloroprocaine), cocaine (cocaine), cocaine (cyclic methyl carbamate), dibucaine (dibucaine), dimethylcarbane (dimethylcarbaine) dimethyl-dicaine, diperoxide, dicyclomine, ecgonidine, ethyl chloride, etidocaine, beta-eucaine, you Puluo octacin, phenethylamine, formoteraine, hexidine, hydroxybutanaine, isobutyl carbamate (isobutyl paminobenzoate), leucinyl mesylate (leucinocaine mesylate), levofloxacin, lidocaine (lidocaine), mepivacaine, methiocarine, chloromethane (methyl chloride), meltecaine (mycecaine), pentanamine ethyl p-aminobenzoate (naepaine), ostacaine (ostacaine), procaine (orthocaine), hydroxyethylcaine (oxazaine), ethoxycaine (paraethoxycaine), fenacaine (phenacaine), phenol (phenol), pipivacaine (picocaine), pyridine caine (picocaine), polidocanol (polidocanol), pramoxine (pramoxine), prilocaine (priocaine), procaine (procaine), propiconazole (procalcitonin), procaine (picocaine), pseudococaine (pseudococaine), pyrrolecaine (pyrdicaine), ropine (rosaine), salicylic alcohol (salicylic acid), bupivacaine (tetracaine), and combinations thereof.

The composition may not include a pharmaceutically active substance selected from the group consisting of proteins, glycosaminoglycans other than hyaluronic acid, and hydroxypropyl methylcellulose.

The composition may be filled in a syringe.

The composition is useful for tissue repair in an individual.

In the composition, the individual may be a mammal. The mammal may be a human, dog, cat, cow, pig, rat or sheep.

The composition may be used for at least one use selected from the group consisting of facial shaping, wrinkle improvement, facial contour surgery, chest shaping, breast augmentation, genitalia enlargement, glans augmentation, urinary incontinence treatment, and arthritis treatment.

The composition may further comprise a pharmaceutically acceptable carrier, excipient, and diluent. The carrier may be, for example, water or a buffer. The buffer may provide a solution with a pH that is nearly unchanged by the addition of the components of the composition. The composition may be an aqueous liquid composition. The composition may be an aqueous buffer composition. The pH of the aqueous buffer composition may be in the physiological pH range, for example between about 6.0 and 8.0. The pH can be adjusted by adding HCl and Na ₂ CO ₃ Or NaOH, or the like. In a specific embodiment, the aqueous buffered composition may include a Phosphate Buffered Solution (PBS). In another specific embodiment, the aqueous buffered composition may comprise Tris (hydroxymethyl) aminomethane (Tris). In some embodiments, additional solutes such as sodium chloride, calcium chloride, and potassium chloride may be added to adjust osmotic pressure and ion concentration.

The composition may be a sterilized composition.

The composition may be included in a container. The container may be a syringe. The composition may be pre-filled into a syringe prior to use. The composition may be administered by a pre-filled syringe.

In another aspect, an apparatus is provided that includes the composition. The device may be a pre-filled syringe. The apparatus may be a sterilization apparatus.

Another aspect provides a kit comprising the prefilled syringe. The kit may include instructions comprising information for administering the composition.

In another aspect, a method of filling tissue in an individual is provided, comprising administering a therapeutically effective amount of the composition to the individual. The method may be used to reinforce, repair or strengthen body tissue or to fill a body cavity. From this point of view, the volume increase (volume augmentation) can be a sustained increase in volume by the components forming the filler composition. The ingredients forming the filler composition may not undergo rapid diffusion. In the methods, "composition" and "individual" are as described above. The administration may be to the skin such as the dermis or within a joint cavity. In the method, the administration may be by syringe, e.g. a pre-filled syringe, to the skin, e.g. intradermal. In the method, the administration may be 0.1ml to 50ml, 0.5ml to 30ml, 0.5ml to 20ml, 0.5ml to 15ml, or 0.8ml to 12ml per administration. In the method, the administration may be 1 administration every 3 months or more, 1 administration every 4 months or more, 1 administration every 5 months or more, 1 administration every 6 months or more, 1 administration every 12 months or more, or 1 administration every 18 months or more.

Hereinafter, the present invention will be described in further detail by way of examples. However, these examples are intended to illustrate the present invention, and the scope of the present invention is not limited thereto.

Detailed Description

Examples

Measurement method

(1) Critical strain (critical strain) measuring method

The critical strain was measured by strain-sweep test (strain-sweep test). Specifically, a DHR-2 rheometer (TA instruments Co.) was run. In the strain sweep test, the instrument temperature was set to 25 ℃, and a geometric body having a diameter of 25 mm was installed to perform correction (calibration). A suitable amount of sample is loaded into the instrument centrally between the upper and lower geometries of the peltier plate. A sufficient amount of sample is overloaded (over-loaded) to prevent starving, and an appropriate amount of sample is loaded so that the residue outside the area under the geometry is not stuck to the sides and top of the geometry even if the residue is removed. After lowering the geometry to the set interval, it is confirmed whether the sample is perfectly filled under the geometry, and then the remaining sample outside the geometry is cut off and removed. Then, the elastic modulus value measured at each strain value when a strain of 0.1% to 1000% was applied to the test piece using the geometry was measured. From the measurement values measured by the rheometer, strain values (Strain%) and elastic Modulus values were obtained, and the Strain values% were converted into logarithms (Strain (%)) by a Transform function built in the program using Graphpad Prism (Prism 8, version 8.4.3 (686)) of Graphpad Software company, and in order to easily confirm the Strain values of the minimum values from the obtained values, the elastic Modulus (Pa) was calculated by a Transform function built in the program with respect to the calculation of the change in value, and then, after the calculation of the graph of the value by the Transform function, the graph was converted into Strain values% of the values before taking the logarithms. From the data obtained by the above procedure, the amount of change in the elastic modulus value with respect to the% of different strain values can be confirmed, and the% of strain value of the minimum elastic modulus value is defined as the critical strain.

(2) Swelling degree measuring method

Hyaluronic acid gel was added to a centrifuge Tube (Conical Tube), and the initial weight was recorded after weighing. After adding 5.0mL of PBS solution (pH 7.4) to the centrifuge tube, vortex and mix well for 1 minute, then seal completely and incubate at room temperature for 24 hours to swell the hyaluronic acid gel. Then, 200. Mu.L of Alxin blue solution was added to the hyaluronic acid gel swelled in the centrifuge tube, vortexed for 1 minute, and then incubated at room temperature for 30 minutes to stain the hyaluronic acid gel. It was centrifuged at 14000g for 10 minutes at 4℃to precipitate a hyaluronic acid gel, and then the supernatant was carefully removed with a pipette. Next, in order to remove the dye of the hyaluronic acid gel, 5.0mL of PBS solution (pH 7.4) was added and vortexed for 1 minute, and then centrifugation was performed again at 14000g for 10 minutes at 4 ℃. After careful removal of the supernatant with a pipette, a further 2 steps of addition of PBS and centrifugation were performed. The swollen hyaluronic acid gel obtained as above was weighed, and the swelling degree was calculated by the following formula:

(3) Average particle size measurement method

Particle size measurements were performed by Microtrac company Particle Size Analyzer S3500. Average particle size measurement was performed by laser refraction analysis after the sample was added to the solvent. The solvent was measured using distilled water. Before execution, the sample inlet was thoroughly washed with distilled water, and then the refractive index of distilled water and the refractive index of the sample were respectively inputted to 1.33 and 1.37, and the type of the sample was set as transparent particles having a random shape. Before measurement, distilled water is filled into a sample inlet of the instrument, and then the sample is diluted by adding an excessive amount of distilled water after being introduced into a tube or the like, and the sample is sufficiently dispersed by using Vortex (Vortex) or the like, and then the instrument is introduced and measurement is performed. In the measurement result value, the average particle size (D50) is taken as data.

(4) Viscoelasticity measuring method

A DHR-2 rheometer (TA instruments Co.) was run. In the viscoelastic test, the instrument temperature was set to 25 ℃, and a geometric body having a diameter of 40 mm was installed to perform correction. A suitable amount of sample is loaded into the instrument centrally between the upper and lower geometries of the peltier plate. A sufficient amount of sample is overloaded to prevent starvation and a suitable amount of sample is loaded that will not stick to the sides and top of the geometry. After lowering the geometry to the set interval, it is confirmed whether the sample is filled under the geometry, and then the remaining sample outside the geometry is cut and removed. Then, shear deformation was periodically applied to the test specimen according to a specific frequency by a certain strain of the geometric body, and the storage modulus and loss modulus values at a frequency of about 0.1Hz among the elastic modulus values thus obtained were defined as elasticity and viscosity, respectively.

(5) Adhesion measurement method

The adhesion was measured as follows. A DHR-2 rheometer (TA instruments Co.) was run. In the viscoelastic test, the instrument temperature was set to 25 ℃, and a geometric body having a diameter of 40 mm was installed to perform correction. A suitable amount of sample is loaded into the instrument centrally between the upper and lower geometries of the peltier plate. A sufficient amount of sample is overloaded to prevent starvation and a suitable amount of sample is loaded that will not stick to the sides and top of the geometry. After lowering the geometry to the set interval, it is confirmed whether the sample is perfectly filled under the geometry, and then the remaining sample outside the geometry is cut off and removed. Then, the force applied to the geometry was measured while setting the geometry interval to a height of 100 μm and pulling it on the lower peltier plate in the vertical axis direction for 180 seconds at a constant speed of 100.0 μm/s. The maximum force is measured at the moment of initial sample detachment due to the adhesion force possessed by the sample between the geometry and the peltier plate, and is defined as the adhesion force.

(6) Compression force measuring method

A DHR-2 rheometer (TA instruments Co.) was run. In the compression force test, the instrument temperature was set to 25 ℃, and a geometric body having a diameter of 25 mm was installed to perform correction. 1mL of the sample was loaded into the center between the upper and lower geometries of the Peltier plate of the instrument. The geometry is lowered to a set interval and then rotated at a low speed so that the sample is centered on the geometry. The force applied to the geometry was then measured while the geometry was lowered from 2500 μm to the 900 μm position at a speed of 13.33 μm/s. When a graph is drawn in which the force measured from the start to the end of the experiment is taken as the Y axis and the time of movement is taken as the X axis, a value corresponding to the lower area value of the graph, which is the graph integrated value, is defined as the compression force.

(7) Injection force measuring method

Injection force was measured using Multitest 2.5-i Universal Testing Machine from Mecmesin. A Load Cell (Load Cell) for measuring the force applied to the instrument is installed and uses a suitable Load Cell having a higher tolerance value than the injection force measurement range. A syringe containing the sample is placed under the instrument load cell and a needle is coupled to the syringe. The push rod with a flat end part is placed on the injector, so that the load unit can apply force uniformly when applying force. After the distance was adjusted to the point where the load cell portion is about to contact the end portion of the push rod, the push rod portion was pressed with the load cell at a speed of 12mm/min, and the applied force was measured. Taking the initial and final portions of the measurement as examples, the pressure value generated when the sample flows into the needle is smaller than the pressure required for injection by the syringe. Further, when the injection of the injected substance is about to end, there is a possibility that the force continues to be applied without the sample, and the applied force is independent of the injection force of the sample. Therefore, after excluding the values of the portion where the sample flows into the injection needle and the corresponding portion near the end of injection, the average of the injection force values measured in the middle portion of the syringe filling liquid after excluding the measured initial injection force value and the late injection force value is defined as the injection force.

Examples 1 and 2: preparation of crosslinked hyaluronic acid

The crosslinked hyaluronic acid having a critical strain of 10% to 100% and a swelling degree of 1% to 100% was prepared as follows.

First, a 1% (w/w) NaOH solution was prepared. 5g of sodium hyaluronate (IV 2.2 to 3.0) was mixed in the prepared 1% NaOH solution to reach 13.00% (w/w), and then stirred to be sufficiently dissolved. Wherein IV refers to the characteristic granularity (intrinsic viscosity). To the above solution, 0.630g of butanediol diglycidyl ether (butanediol diglycidylether, BDDE) (Sigma-Aldrich) was added and further stirred to uniformly mix them, and after taking out, a crosslinking reaction was performed at 40℃for 16 to 20 hours, thereby preparing a crosslinked hyaluronic acid gel.

Next, the resulting crosslinked gel was put into a dialysis membrane and sealed, and then dialysis was performed using 1mol/kg NaCl solution and 1 XPBS aqueous solution as dialysis solutions, thereby removing unreacted crosslinking agent. After the dialysis was completed, the content was corrected with 1×pbs until the final sodium hyaluronate concentration removed reached 18mg/mL, taking into account the loss rate of the weight of sodium hyaluronate initially charged. At this time, the lidocaine hydrochloride hydrate content was corrected so as to reach 3mg/mL. As a result, a PBS solution containing 18mg/mL of crosslinked hyaluronic acid and 3mg/mL of lidocaine was prepared. The prepared crosslinked hyaluronic acid-containing solution was pulverized using a stirrer (Retsch GM-200) at 8000rpm for 180 seconds. Then, 1ml of the crushed crosslinked hyaluronic acid-containing solution was filled into a glass syringe and then subjected to high-temperature steam sterilization. The material obtained by the above procedure was designated as example 1.

The same preparation as in example 1 was conducted except that the crosslinked hyaluronic acid-containing solution was pulverized with a stirrer (Retsch GM-200) at 5000rpm for 75 seconds, taking example 2 as an example.

Comparative example 1

The preparation was performed in the same manner as in example 1 except that the crosslinked hyaluronic acid-containing solution was pulverized with a stirrer (Retsch GM-200) at 2000rpm for 180 seconds, as an example of comparative example 1.

Comparative example 2 and comparative example 3:

comparative example 2 used Juvederm Voluma Lidocaine from Allergan as single-phase crosslinked hyaluronic acid, and comparative example 3 used Restylane Lyft Lidocaine from Galderma as double-phase crosslinked hyaluronic acid.

Experimental example 1: measurement of physical Properties of crosslinked hyaluronic acid gel

Physical properties of the crosslinked hyaluronic acid gel were measured by the methods described in the measurement methods (1) to (7) of the above examples, and the results thereof are shown in table 1 below.

TABLE 1

As shown in Table 1, it was confirmed that the swelling degree of example 1 and example 2 was significantly decreased compared with comparative example 2 (Juvederm Voluma L, allergan) as a conventional single-phase crosslinked hyaluronic acid filler. Taking comparative example 3 (Restylane Lyft L, galderm) as an existing biphasic crosslinked hyaluronic acid filler as an example, the degree of swelling was not measured because the crosslinked hyaluronic acid gel and the solution could not be separated when the degree of swelling was measured.

From the results of the physical properties shown in Table 1, it was confirmed that examples 1 and 2 have similar compressive force and adhesive force to those of the conventional single-phase filler, and therefore have a low possibility of detachment from the injection site and a low degree of swelling, and thus the swelling phenomenon is remarkably reduced when injected into the body. Furthermore, it is known from the fact that it has a high elastic modulus (G'), which is capable of maintaining the injection shape for a long period of time.

Experimental example 2: the effect on lift and volume increase when cross-linked hyaluronic acid was applied to animals was measured: lifting and volume increase test (lifting and volumizing test)

52 female (20.+ -.3 g) nude mice (SKH 1-hr) of six weeks old were introduced from oriientbio corporation (Korea) and acclimatized for about 1 week or more. After intraperitoneal injection of ketamine (100 mg/kg) and Long Peng (10 mg/kg) mixed anesthetics into the above-mentioned domesticated mice, the above-mentioned examples 1 to 2 and comparative examples 1 to 3 were subcutaneously injected with 0.1mL, respectively, in the back area using a glass syringe. The body weight of the individual was measured on the indicated date after injection. The height of the injection site, the maximum length of the injectate and the volume were measured for the injection site using Primos 5.8E (Canfield Sc ientific Inc, NJ, USA). In addition, visual observations were made and photographs were taken for the injection site.

Fig. 1 is the change in volume observed up to 42 weeks after intradermal injection of the hyaluronic acid gel. As shown in fig. 1, taking comparative example 1 as a conventional single-phase crosslinked hyaluronic acid gel as an example, the initial volume of injection was greatly increased (about 100% increase from the initial injection volume) and the volume was reduced in response to the decrease over time, whereas examples 1 and 2 had little or no volume change (about 10% increase from the initial injection volume) compared to the initial injection. Further, it was confirmed that the volume loss of example 1 and example 2 was relatively small over the course of 24 weeks.

From the above, it was confirmed that examples 1 and 2 have little swelling phenomenon at the initial stage of injection, can hold volume at the time of injection, and have little change in volume from after injection to 24 weeks, and thus have excellent in vivo persistence and in vivo compatibility.

Claims

1. A hyaluronic acid cross-linked body having a critical strain of 10% to 100% and a swelling degree of 1% to 100%.

2. The crosslinked hyaluronic acid according to claim 1, wherein,

the average particle size thereof is in the range of 50 μm to 150. Mu.m.

3. The crosslinked hyaluronic acid according to claim 1, wherein,

the elastic modulus (G') thereof is 400Pa to 2000Pa.

4. The crosslinked hyaluronic acid according to claim 1, wherein,

the viscous modulus (G') is from 100Pa to 600Pa.

5. The crosslinked hyaluronic acid according to claim 1, wherein,

the compression force is 15 N.s to 100 N.s.

6. The crosslinked hyaluronic acid according to claim 1, wherein,

the injection force is 1N to 50N.

7. The crosslinked hyaluronic acid according to claim 1, wherein,

which is crosslinked by a crosslinking agent having difunctional epoxy groups.

8. The crosslinked hyaluronic acid according to claim 7, wherein,

the crosslinking agent having a difunctional epoxy group is at least one selected from the group consisting of 1, 4-butanediol diglycidyl ether, polyethylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, polytetramethylene glycol diglycidyl ether, polyglycerol polyglycidyl diether, glycerol diglycidyl ether, triethylenediglycidyl ether, trimethylolpropane triglycidyl ether, ethylene diglycidyl ether, neopentyl glycol diglycidyl ether, and 1, 6-hexanediol diglycidyl ether.

9. A filler composition comprising the hyaluronic acid cross-linked body of any of claims 1-8.

10. The filler composition of claim 9, wherein,

the concentration of hyaluronic acid cross-linked body is 10mg/mL to 30mg/mL.

11. The filler composition of claim 9, wherein,

it does not further comprise uncrosslinked hyaluronic acid.

12. The filler composition of claim 9, further comprising a local anesthetic.

13. The filler composition of claim 9, wherein,

which fills the syringe.

14. The filler composition of claim 9, wherein,

the composition is used for at least one use selected from the group consisting of facial shaping, wrinkle improvement, facial contour surgery, chest shaping, breast augmentation, genital enlargement, glans augmentation, urinary incontinence treatment, and arthritis treatment.